PureCube Maleimide Activated Agarose is available as off-the-shelf item and as a bulk product. Our careful in-house production provides low lot-to-lot variation, making activated agaroses also suitable for commercial production. PureCube Maleimide Activated Agarose carries a maleimide functionality. Proteins bind covalently and irreversibly to free thiol groups.
PureCube Maleimide Activated Agarose provides:
Activated Maleimide agarose resins from Cube Biotech were successfully used in the following publications and patents:
|cap-specific nucleoside 2′-O-methyltransferase.
||Roos T., Yazdan Oanah B., Conzelmann M., Thess A., Buob D., Kunze M., Wagner V.2016-August-121
|Recombinant mutant PPases
||Kunze M., Hadler F.N., Yazdan Panah B.,Ross T. 2
||Mahon C.S., Wildsmith G.C., Haksar D., de Poel E., Beekman J.M., Pieters R.J., Webb M.E., Turnbill W.B.3
||Coupling proteins to via their activated thiol groups
||Affinity to thiol groups
||up to 6 mL/min
||Delivered as a 50 % suspension
||Affinity agarose with activated Maleimide groups
- Coupling Buffer I (PBS) pH 7.2, 250 mL
- Coupling Buffer II pH 6.5, 250 mL
- Storage Buffer I, pH 6.5, 250 mL
- Storage Buffer II, pH 7.5, 250 mL
- Centrifuge for 15 mL tubes
- Centrifuge tubes (15 mL)
- End-over-end mixer or thermomixer
|A.||Maleimide coupling protocol|
- Transfer 1 mL PureCube Maleimide-Activated Agarose suspension (corresponding to 1 mL bed volume) into a 2 ml microcentrifuge tube.Tip: The coupling reaction can be linearly scaled up and down, by increasing or decreasing the amounts of buffers and solutions described in this protocol.
- Spin the tube at 500 x g to pellet the agarose. Remove the supernatant.
- Wash the Activated Agarose three times with 1 ml coupling buffer I or II. Tip: Choose coupling buffer I or II depending on the pH stability of your protein. Use the same coupling buffer throughout the procedure.
- Resuspend 1-3 mg protein in 500 µl coupling buffer I or II.
- Add the protein to the activated agarose and mix by vortexing. Tip: When coupling a particular protein for the first time, try 3-5 different protein concentrations to make sure you are offering enough protein in the reaction but not wasting any protein.
- Depending on the temperature stability of the protein, incubate at 20°C for 2 hours or at 4° C overnight on an endover-end shaker or thermoshaker.
- Spin the tube at 500 x g to pellet the agarose. Remove the supernatant and analyze the supernatant in a spectrophotometer. Record absorption at 280 nm to monitor coupling efficiency.
- Wash the agarose three times with 1 ml coupling buffer and once with 1 ml double distilled water.
- Tip: Monitoring absorbance at 280 nm tells you about the coupling efficiency of the protein (compare A280 of the original protein solution to the supernatant in step 6 to determine % coupling). It also helps you identify the optimal amount of protein required for efficient coupling.
- Resuspend the coupled Agarose in 1 mL Storage buffer I or II, yielding a 50% suspension. Store at 4°C.
1. Roos T., Yazdan Oanah B., Conzelmann M., Thess A., Buob D., Kunze M., Wagner V. inventor; Method for adding cap structures to rna using immobilized enzymes. International patent: WO2016193226A1. 2016-December-08
2. Kunze M., Hadler F.N., Yazdan Panah B.,Ross T. inventor; CureVae AG, Tübingen, Immobilized Inorganic Pyrophosphatase (Ppase), Us-Patent: US 2019 / 0177714 A1; September-21-2018
3. Mahon S. et al. (2019). A ‘catch-and-release’ receptor for the cholera toxin. Faraday Discussions. 10.1039/C9FD00017H.